This thesis uses surface sensitive tools to characterize the effect of a solid surface on immobilized biomacromolecules. This includes understanding how the surface can change the affinity of these macromolecules to small molecules compared to bulk studies. Two classes of immobilized biomacromolecules, the supported lipid bilayer (SLB) and the Lac repressor protein (LacI), are characterized using microcantilever sensors and quartz crystal microbalance with dissipation (QCM-D). The first part of this thesis reports the use of microcantilever beams, an ultrasensitive sensor for measuring the surface free energy changes on a substrate induced by molecular adsorptions, to probe the interaction between a solid surface and a phospholipid bilayer. This sensing method integrates two well-developed techniques: solid-supported lipid bilayers (SLBs) and the microcantilever (MC) sensors. Studying the adsorption free energy of lipid bilayers on a solid surface allows better characterizing of the formation and stability of SLBs. Microcantilever converts the Gibbs free energy change taking place on its surface into a mechanical deformation. As molecules physisorb or chemisorb onto the surface of the microcantilevers, the microcantilevers bend, either due to induced compressive or tensile stresses, which result from the surface free energy change. By monitoring the deflection values of the microcantilevers, the real-time surface free energy change during the SLB formation can be detected. This thesis has led to the development of a novel biosensor--lipid membrane coated microcantilevers--to detect the adsorption, insertion, aggregation and solubilizing effect of membrane-active substances, such as surfactants and peptides, on the phospholipid membranes. To better characterize the surface free energy, SLBs doped with charged lipids or cholesterol are shown to alter the surface free energy. We can predict this change in surface free energy using a thermodynamic model. Application of this membrane-coated cantilever is put into use for detecting how amphiphilic molecules interact with SLBs, as well as for probing the abrupt conformational change of SLBs during a temperature induced phase-transition. This study systematically demonstrates various usage aspects of microcantilever to characterize the SLBs, and how this technique may advance the biophysical knowledge of the lipid membrane, one of the essential building blocks of life. The second part of this thesis reports the use of both microcantilever sensors and QCM-D to measure the adsorption free energy and mass of a model protein, the Lac repressor (LacI), and compare how a modified T334C mutant that includes a cysteine group to orient the protein on the gold surface through a covalent sulfur bonds retains its binding capabilities over that of wild type LacI. The main challenge of this work is to unravel how the adsorption of biomacromolecules at the solid/liquid interface leads to surface free energy changes and ultimately changes the stress of the underlying solid surface (the cantilever). The uses of microcantilever sensors and QCM to probe the interactions that take place on SLBs and surface-bound proteins have the advantage of being a sensitive, real-time, and label-free technique.

The primary objective of this study was to develop a sensitive and cost-effective sorption system to analyze adsorption and diffusion of different gases on micro porous materials and nanotubes. A high pressure Quartz Crystal Microbalance (QCM) based adsorption apparatus for single-component gas was developed. A QCM is an acoustic-wave resonator in which the acoustic wave propagates through the crystal. Therefore, it is highly responsive to addition or removal of small amounts of mass adsorbed or deposited on the surface of the crystal. This mass sensitivity makes the QCM an ideal tool for the study of gas adsorption. The QCM-based adsorption apparatus is advantageous over the commercialized none-gravimetric and gravimetric equipment in a way that it is low-cost, highly sensitive and accurate for mass sorption applications, satisfactorily stable in a controlled environment, and can be used for thin films. The high pressure apparatus was calibrated using Matrimid 5218, whose thermodynamic properties and adsorption parameters are known. The Matrimid was spin-coated onto a 14 mm-diameter QCM, and sorption equilibrium data for were obtained for CO₂ gas at 25, 30, 48, and 52 ºC and partial pressure range between 0 to 4 bar. In order to compare the experimental data with available literature data, the experimental data was fitted into a dual-mode adsorption model. The model results from Henry's law and a Langmuir mechanism. Comparison of the experimental adsorption isotherm of Matrimide for CO₂ gas with literature data showed reasonable agreement between the experimental and literature data. In this study, the adsorption parameters of aluminosilicate nanotubes are observed. Aluminosilicate nanotubes are ideal materials for chemical sensing, molecule separation, and gas storage; hence, there is a need for adsorption and diffusion data on this material. The adsorption of CO₂, N₂, and CH₄ gases on aluminosilicate nanotubes samples has been studied in the temperature range of 20° to 120° Celsius and pressure range of 0 to 8 bar. The experimental results yield the CO₂ and N₂ heat of adsorptions of -32.9 and -28.1 kJ/mol respectively.

The contamination of drinking water by microbial pathogens is recognized as one of the most pressing water supply problems of our day. To minimize the impact of pathogens and parasites on the environment and public health, accurate methods are needed to evaluate their presence and concentration. Although various techniques exist to detect certain pathogens in water (e.g., immunofluorescence or PCR techniques), these are time- and labor-intensive. A direct, real-time method for detection and quantification of target organisms would thus be very useful for rapid diagnosis of water safety. A quartz crystal microbalance with dissipation monitoring (QCM-D) based biosensor for detection of waterborne pathogens (i.e., Escherichia coli O157:H7) was developed. The detection platform is based on the immobilization of affinity purified antibodies onto gold coated QCM-D quartz crystals via a cysteamine self-assembled monolayer. The results show that the optimal sensor response is the initial slope of the dissipation shift. A highly log-log linear response is obtained for detection of E. coli O157:H7 over a broad range of cell concentration from 3 x 105 to 1 x 109 cells/mL. The prepared biosensor also exhibits a log-log linear working range from 107 to 109 cells/mL for E. coli K12 D21, a non-pathogenic model organism. The biosensor also shows satisfactory selectivity using Bacillus subtilis . To our knowledge, this is the first study demonstrating the use of the slope of the dissipation shift as a sensor response when using QCM-D technology. / Keywords: Biosensor, QCM-D, E. coli O157:H7, polyc1onal antibodies, dissipation slope, cysteamine, self-assembled monolayer

Thesis (M.S.)--University of Texas at El Paso, 2008. / Title from title screen. Vita. CD-ROM. Includes bibliographical references. Also available online.

Thesis (M.For.Sc.)--University of Western Australia, 2007.

Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 Jan 27

The biomedical application of nanotechnology has come into the spotlight, with the promise of ‘personalised’ therapeutics that couple early diagnosis with targeted therapeutic activity. Due to the rapid growth of the biomedical applications of nanoparticles, along with the lack of understanding concerning their interactions with biomolecules, there is a pressing need for the development of standard methods directed at investigating the effect of introducing these unique particles into the human body. The central aim of this research is to establish a platform directed at assessing the biological fate of pioneering therapeutic particulate agents, such as metallophthalocyanines (MPcs) and multi-walled carbon nanotubes (FMWCNTs). In particular, we proposed, that Quartz Crystal Microbalance with Dissipation (QCM-D) technology may be employed to assess the composition of blood protein corona deposited on the therapeutic surface, and subsequently assess the biocompatibility of such particles. The proposed method of protein detection utilises the nanogram sensitivity of QCM-D technology to monitor highly specific antibody-antigen interactions. In particular those interactions which occur when probe antibodies are used to detect adsorbed blood proteins deposited on target particle-modified sensor surfaces. Protein detection analysis was directed toward identification of surface bound human serum albumin, complement factor C3c, and human plasma fibrinogen. Preliminary analysis of generic biomedical surfaces indicated human serum albumin demonstrates a higher binding affinity towards positively charged surfaces (i.e. cysteamine self-assembled monolayer), followed by hydrophobic surfaces. Detection of complement C3c, corresponded with literature, where lower levels were detected on negatively charged surfaces (i.e. mercapto undecanoic acid self-assembled monolayer), and higher levels of more hydrophobic surfaces (i.e. 11-amino undecane thiol self-assembled monolayer). Human plasma fibrinogen was observed to favour hydrophilic over hydrophobic self-assembled monolayer surfaces, which was in accordance with literature. Application of the proposed protein detection method for biocompatibility analysis of target therapeutic molecules, namely metallophthalocyanines and acid functionalised multi-walled carbon nanotubes, demonstrated a dependence on modified-surface film characteristics, such as surface charge and topography with regards to human serum albumin and human plasma fibrinogen analysis representing new insights into their potential biomolecular interactions The highest levels of detected human serum albumin and complement C3c were detected on the GePcSmix-modified surfaces. AlPcSmix-modified surfaces analysis suggested the highest levels of human plasma fibrinogen. Two methods of acid functionalisation were employed, using both nitric and sulphuric acid, and pure nitric acid. A general increase in detected human serum albumin, corresponding with an increase in functionalisation time, was observed. Complement C3c detection suggested an increase in deposited complement C3c, with increasing functionalisation time, when assessing nitric acid functionalised multi-walled carbon nanotubes, and a decrease, with increasing functionalisation time, when assessing nitric and sulphuric acid functionalised multi-walled carbon nanotubes. Analysis of human plasma fibrinogen was inconclusive, as were cytotoxicity experiments utilising MCF-7 cells in the presence of metallophthalocyanine complexes, raising simultaneously important considerations for their application and study. In the first such detailed examination of its kind it was concluded that the proposed method of protein detection, using QCM-D, allows for the rudimentary but rapid means of analysis of select protein corona deposited on particulate biomedical surfaces.

This PhD project aimed to investigate the possibility of using Odorant Binding Proteins (OBPs) as sensing layers of chemical sensors, for the detection of organic compounds in both vapour and liquid phases. OBPs are small soluble proteins present in high concentrations in the olfactory system of vertebrates and insects. OBPs are attractive in the biosensor field since they can bind odorants and pheromones in a reversible way. They are resistant to high temperatures and protease activity and they can be easily expressed in large amounts. OBPs belonging to different species of mammals and insects were utilised for developing biosensors relied on different transduction mechanisms. Recombinant OBPs were grafted on the gold electrode of transducers by using Self-assembled monolayers (SAMs) of alkanethiols. The efficiency of the immobilisation method was proved by using electrochemical techniques. Quartz crystal microbalances (QCMs), screen-printed electrodes (SPEs) and interdigitated electrodes (IDEs) were employed for developing three types of OBP-based biosensors. I. QCMs functionalised with OBPs were tested against pheromones (i.e. bombykol and bombykal) and volatile compounds found in foodstuffs (i.e. pyrazine derivatives and geosmin) in vapour phase. The QCM based biosensors showed a good degree of selectivity and a detection limit of the order of parts per billion, in air. II. In liquid phase, impedimetric biosensors based on SPEs also showed a good selectivity and sensitivity being able to detect analyte concentrations of the order of 10-9 M. III. OBPs immobilised on the gold electrodes of IDEs were instead tested against S-(+) carvone vapour, proving that the binding activity of the proteins was preserved in vapour phase and can be quantified as variation of capacitance. The developed OBP biosensors showed good selectivity, sensitivity and stability over time in both liquid and vapour phase. The responses of the sensors were reversible, allowing to the device to be used several times. Moreover, the biosensors were label-free, hence the interaction between OBPs and ligand was directly detected without using auxiliary probes/species. With these findings, we envisage the use of our biosensors in several applications, including monitoring of the quality of food along the transportation and storage, controlling of pests and useful insects in agriculture, or as analytical devices for studying the dynamics in binding processes.

The electrochemical quartz crystal microbalance (EQCM) has been proven an effective mean of monitoring up to nano-scale mass changes related to electrode potential variations at its surface. The principles of operation are based on the converse piezoelectric response of quartz crystals to mass variations on the crystal surface. In this work, principles and operations of the EQCM and piezo-electrodes are discussed. A conductive oxide, ruthenium oxide (RuO2) is a promising material to be used as a diffusion barrier for metal interconnects. Characterization of copper underpotential deposition (UPD) on ruthenium and RuO2 electrodes by means of electrochemical methods and other spectroscopic methods is presented. Copper electrodeposition in platinum and ruthenium substrates is investigated at pH values higher than zero. In pH=5 solutions, the rise in local pH caused by the reduction of oxygen leads to the formation of a precipitate, characterized as posnjakite or basic copper sulfate by means of X-ray electron spectroscopy and X-ray diffraction. The mechanism of formation is studied by means of the EQCM, presenting this technique as a powerful in-situ sensing device.

No description available.